Antigen presenting cells (APCs) of the immune system, when pulsed with tumor-derived GRP94 and calreticulin chaperone proteins, are able to stimulate an immune response specific to the tumor of origin. This response is mediated by tumor-specific peptides bound to GRP94 and calreticulin. To determine how chaperone/peptide complexes elicit this immune response, studies will focus on the intracellular trafficking pathways by which chaperone/peptide complexes are processed by antigen presenting cells First, surface binding and internalization of fluorescent chaperone/peptide complexes by macrophages and dendritic cells at different stages of development and activation will be studied using high-resolution fluorescence confocal microscopy and FACS analysis. Next, utilizing fluorescent as well as electron microscopy, the mechanism of chaperone/peptide complex uptake and the subcellular fate of internalized chaperones will be investigated. Finally, by using existing monoclonal antibodies specific for particular peptide/MHC complexes, the point of formation and subcellular trafficking pathways of peptide/MHC complexes determined. The combined results of these three lines of investigation will provide substantial insights into how extracellular antigenic peptide/chaperone complexes are trafficked and processed by APCs.